Establishment Of Breast Cancer Mouse Model And The Subtypes Analyze Of Bone Marrow MDSC Cells

Breast cancer is one of the malignant tumors that cause the high incidence and mortality of cancer in the world.As the role of Myeloid-derived suppressor cells(MDSCs)in tumorigenesis,progression,and metastasis has been gradually demonstrated,so the research on MDSCs cell received more and more consideration.However,due to the heterogeneity and the complex biological functions of MDSCs and the existing cell phenotype for its identification can not clearly delineate its boundaries with other myeloid-derived cells,such as tumor-associated macrophages(TAM),Tumor-associated neutrophils(TAN),cells with similar inhibitory functions,and granulocytes,monocytes,dendritic cells,and so on.Therefore,the study of molecular mechanisms about how MDSCs work is limited.Objectives:To analyze the expression of chemokine receptors on the surface of the 4T1 cell line and the chemokine receptor and Toll-like receptor(TLR)on the surface of bone marrow MDSCs in transplanted mouse,the chemokine receptor and Toll-like receptor surface markers map of bone marrow MDSCs cell subunits in 4T1 cells from 4T1 transplanted mouse were established,in order to provide prerequisites for further researches about molecular mechanisms of different subtypes.Methods:The 4T1 transplanted mouse model was constructed by orthotopic transplantation.The expression of surface chemokine receptor in 4T1 cell line was analyzed by flow cytometry.Flow cytometry was used to analyze chemokine receptors and Toll-like receptors on the surface of bone marrow MDSCs in normal and transplanted mice.The subtypes of bone marrow MDSCs were analyzed by multicolor flow analysis.Results:The model of transplanted tumor of 4T1 cells was established and the time curve of tumor development in mice was obtained.The expression of chemokine receptors on the surface of 4T1 cells showed that the positive expression rates of CCR1,CCR5,CCR7,CCR9 and CXCR7 on the surface of 4T1 cells were more than 25%,while the other chemokine receptor expression was very low,almost no expression.Through the mouse bone marrow MDSCs cell surface chemokine receptor and Toll-like receptor for analysis,based on the results obtained from the preliminary analysis,the subtypes of bone marrow MDSC cells were analyzed by CCR2,CXCR2,CXCR4,CXCR6 and CX3CR1 for further multicomponent(8-color)flow cytometry analysis.CCR2,CXCR2,CXCL4,CXCR6,CX3CR1 were incubated with other seven-color antibodies to further analyze the subtypes of bone marrow MDSC cells.30 different bone marrow cell subtypes were identified.Conclusion:Metastatic mouse breast cancer model was successfully established by orthotopic transplantation of tumor,the expression of chemokine receptor on the surface of 4T1 cell line was successfully analyzed by flow cytometry,and the chemokine receptor and Toll-like receptor on the surface of mouse bone marrow MDSCs were successfully analyzed,what's more,and multicolor analysis of bone marrow MDSC cell subtypes was performed.With this series of experiments,we can more clearly understand the migration of tumor and immune cells,and how does MDSCs promote tumor escaping.At the same time,it offers new target and orientation for tumor therapy.